Peptide nucleic acid probes for analysis of certain staphlococcus species

ABSTRACT

The present invention relates to peptide nucleic acid (PNA) probes, PNA probe sets and methods for the analysis of certain  Staphylococcus  species optionally present in a sample. The invention further relates to diagnostic kits comprising such PNA probes or PNA probe sets.

RELATED APPLICATIONS

This application claims the benefit, as a continuation-in-part, of U.S.Provisional Application Ser. No. 60/525,591, filed Nov. 26, 2003; PCTApplication No. PCT/US2004/039781, filed Nov. 24, 2004; and U.S.application Ser. No. 10/580,727, filed May 24,2006. The entire contentof these applications are incorporated herein by this reference in theirenterity.

FIELD OF THE INVENTION

The present invention relates to peptide nucleic acid (PNA) probes, PNAprobe sets and methods for the analysis of certain Staphylococcusspecies optionally present in a sample. The invention further relates todiagnostic kits comprising such PNA probes or PNA probe sets. Themethods and kits of the invention are particularly useful forsimultaneous analysis of one or more of Staphylococcus species otherthan S. aureus and of S. aureus.

BACKGROUND

Staphylococcus aureus is a well-known human pathogen associated withsurgical site infections, bloodstream infections (BSI) and other seriousinfections. In contrast, other Staphylococcus species, such asparticularly Staphylococcus epidermidis, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus lugdenensis, Staphylococcussaprophyticus and Staphylococcus simulans commonly found on the skin,are with the exception of catheter-related infections rarely of clinicalsignificance. Furthermore, in those instances where a BSI is caused by aStaphylococcus species other than S. aureus there is often a differencein antibiotic susceptibility or viability between those organisms and S.aureus. Differentiation between Staphylococcus species other than S.aureus and S. aureus is therefore extremely important for directingappropriate patient therapy and patient management.

The presence of Staphylococcus species in clinical specimens isroutinely determined by the presence of Gram-positive cocci in clusters(GPCC) by Gram staining and microscopic analysis, however;differentiation between S. aureus and other Staphylococcus species mustawait subculture, overnight incubation followed by biochemical analysis,such as coagulase and latex agglutination testing, or more recently bymolecular testing. The presence of other Staphylococcus species is oftendetermined on the basis of a negative tube coagulase test result and istypically reported as coagulase-negative staphylococci (CNS). This isparticularly problematic for analysis of GPCC positive blood culturebottles as only 20-30% of GPCC positive blood bottles are due to S.aureus (Karlowsky et al., Ann Clin Microbiol Antimicrob 3:7 (2004)). Inthe majority of cases physicians therefore have to make decisions basedon negative test results, i.e. absence of S. aureus.

DNA probes for analysis of Staphylococcus aureus and all Staphylococcusspecies (genus-specific probes) have been described (WO0066788, Kempf etal., J. Clin. Microbiol 38:830-838 (2000)) as well as PNA probes for theanalysis of S. aureus (U.S. Pat. No. 6,664,045, Oliveira et al., J.Clin. Microbiol. 40:247-251 (2002)). These probes all target sequencesthat are either species-specific or genus-specific.

Comparative analysis of ribosomal RNA (rRNA) sequences or genomic DNAsequences corresponding to said rRNA (rDNA) has become a widely acceptedmethod for establishing phylogenetic relationships between bacterialspecies (Woese, Microbiol. Rev. 51:221-271 (1987)). Consequently,Bergey's Manual of systematic bacteriology has been revised based onrRNA or rDNA sequence comparisons. Ribosomal RNA or rDNA sequencedifferences between closely related species enable design ofspecies-specific probes for microbial identification thus enablingdiagnostic microbiology to be based on a single genetic marker ratherthan a series of phenotypic markers as in traditional microbiology(Delong et al., Science 342:1360-1363 (1989)). However, the design ofprobes targeting a cohort of species is particularly problematic andrequires a combination of highly specific probe constructs and uniquetarget sequences.

PNAs are useful candidates for investigation when developing probestargeting a subset of species because they hybridize to nucleic acidswith increased sequence specificity as compared to DNA probes. Prior arttherefore also comprises examples of PNA probes targeting mycobacteriaother than Mycobacterium tuberculosis (U.S. Pat. No. 6,753,421) andenterococci other than Enterococcus faecalis (Oliveira et al., Abstract#D-2003, lnterscience Conference on Antimicrobial Agents andChemotherapy, Sept. 27-30, 2002, San Diego, Calif.).

Despite its name, Peptide Nucleic Acid (PNA) is neither a peptide nor anucleic acid, it is not even an acid. PNA is a non-naturally occurringpolyamide that can hybridize to nucleic acids (DNA and RNA) withsequence specificity according to Watson-Crick base paring rules (See:U.S. Pat. No. 5,539,082) and Egholm et al., Nature 365:566-568 (1993)).However, whereas nucleic acids are biological materials that play acentral role in the life of living species as agents of genetictransmission and expression, PNA is a recently developed totallyartificial molecule, conceived in the minds of chemists and made usingsynthetic organic chemistry. PNA also differs structurally from nucleicacid. Although both can employ common nucleobases (A, C, G, T, and U),the backbones of these molecules are structurally diverse. The backbonesof RNA and DNA are composed of repeating phosphodiester ribose and2-deoxyribose units. In contrast, the backbones of the most common PNAsare composed on (aminoethyl)-glycine subunits. Additionally, in PNA thenucleobases are connected to the backbone by an additional methylenecarbonyl moiety. PNA is therefore not an acid and therefore contains nocharged acidic groups such as those present in DNA and RNA. Thenon-charged backbone allows PNA probes to hybridize under conditionsthat are destabilizing to DNA and RNA. Such attributes enable PNA probesto access targets, such as highly structured rRNA and double strandedDNA, known to be inaccessible to DNA probes (See: Stephano &Hyldig-Nielsen, IBC Library Series Publication #948. InternationalBusiness Communication, Southborough, Mass., pp. 19-37 (1997)). PNAprobes are not the equivalent of nucleic acid probes in structure orfunction.

Positive identification of Staphylococcus species other than S. aureuswould be advantageous in many cases and simultaneous analysis of both S.aureus and other Staphylococcus species would be ideal as treatmentdecisions for the presence of either S. aureus or other Staphylococcusspecies would always be based on a positive test results. This featurewould also offer a significant advantage when a mixture of S. aureus andother Staphylococcus species are present.

SUMMARY OF THE INVENTION

This invention is directed to PNA probes, or PNA probe sets and theiruse as well as kits useful for the analysis of certain Staphylococcusspecies, preferably those other than Staphylococcus aureus optionallypresent in a sample of interest. In accordance with the claims, the PNAprobes are directed to ribosomal RNA (rRNA) or the genomic sequencescorresponding to said rRNA (rDNA) or its complement.

In one embodiment, this invention is directed to PNA probes for analysisof one Staphylococcus species other than S. aureus, such as but notlimited to Staphylococcus epidermidis, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus lugdenensis, Staphylococcussaprophyticus and Staphylococcus simulans.

In another embodiment, this invention is directed to PNA probes foranalysis of two or more Staphylococcus species other than S. aureus.

In one aspect, provided herein are PNA probes comprising a nucleobasesequence suitable for the analysis of one or more Staphylococcus speciesother than S. aureus.

In one aspect, provided herein are PNA probes comprising a nucleobasesequence suitable for the analysis of two or more Staphylococcus speciesother than S. aureus.

In another embodiment, a target sequence of the Staphylococcus speciescomprises rRNA, rDNA or a complement of rRNA or rDNA.

In one embodiment, the nucleobase sequence suitable for the analysis ofStaphylococcus epidermidis, comprises a PNA probe complementary to atarget sequence of Staphylococcus epidermidis rRNA or rDNA or itscomplement.

In another embodiment, the nucleobase sequence suitable for the analysisof Staphylococcus simulans, comprises a PNA probe complementary to atarget sequence of Staphylococcus simulans rRNA or rDNA or itscomplement.

In one embodiment, the nucleobase sequence suitable for the analysis ofStaphylococcus haemolyticus, comprises a PNA probe complementary to atarget sequence of Staphylococcus haemolyticus rRNA or rDNA or itscomplement.

In another embodiment, the nucleobase sequence suitable for the analysisof Staphylococcus lugdunensis, comprises a PNA probe being complementaryto a target sequence of Staphylococcus lugdunensis rRNA or rDNA or itscomplement.

In one embodiment, the nucleobase sequence suitable for the analysis ofStaphylococcus saprophyticus comprises a PNA probe complementary to atarget sequence of Staphylococcus saprophyticus rRNA or rDNA or itscomplement.

In one embodiment, the nucleobase sequence suitable for the analysis ofStaphylococcus epidermidis and one or more other Staphylococcus speciesother than Staphylococcus aureus, comprises a PNA probe complementary toa target sequence of Staphylococcus epidermidis rRNA or rDNA or itscomplement.

In another embodiment, the nucleobase sequence suitable for the analysisof Staphylococcus simulans and one or more other Staphylococcus speciesother than Staphylococcus aureus, comprises a PNA probe complementary toa target sequence of Staphylococcus simulans rRNA or rDNA or itscomplement.

In one embodiment, the nucleobase sequence suitable for the analysis ofStaphylococcus haemolyticus and one or more other Staphylococcus speciesother than Staphylococcus aureus, comprises a PNA probe complementary toa target sequence of Staphylococcus haemolyticus rRNA or rDNA or itscomplement.

In one embodiment, the nucleobase sequence suitable for the analysis ofStaphylococcus lugdunensis and one or more other Staphylococcus speciesother than Staphylococcus aureus, comprises a PNA probe complementary toa target sequence of Staphylococcus lugdunensis rRNA or rDNA or itscomplement.

In another embodiment, the nucleobase sequence suitable for the analysisof Staphylococcus saprophyticus and one or more other Staphylococcusspecies other than Staphylococcus aureus, comprises a PNA probecomplementary to a target sequence of Staphylococcus saprophyticus rRNAor rDNA or its complement.

In one embodiment, at least a portion of the probe is at least about 86%identical to the nucleobase sequence or complement thereof selected fromthe following sequences: AGA-CGT-GCA-TAG-T (Seq. Id. No. 1),GCT-AAT-ACG-GCG (Seq. Id. No. 2), GCT-AAT-ACG-CCG-C (Seq. Id. No. 3).

In another embodiment, at least a portion of the probe is selected fromthe following sequences: AGA-CGT-GCA-TAG-T (Seq. Id. No. 1),GCT-AAT-ACG-GCG (Seq. Id. No. 2), GCT-AAT-ACG-CCG-C (Seq. Id. No. 3).

In one embodiment, the probe sequence is between about 8-17 subunits inlength.

In one embodiment, the probe is labeled with at least one detectablemoiety.

In one embodiment, the detectable moiety or moieties are selected fromthe group consisting of: a conjugate, a branched detection system, achromophore, a fluorophore, a spin label, a radioisotope, an enzyme, ahapten, an acridinium ester and a luminescent compound.

In one embodiment, the probe is self-reporting. In another embodiment,the probe is a PNA Linear Beacon. In one embodiment, the probe isunlabeled. In one embodiment, the probe is bound to a support. Inanother embodiment, the probe further comprises a spacer or a linker.

In one embodiment, in situ hybridization is used for analysis of one ormore Staphylococcus species other than S. aureus.

In one aspect, provided herein are PNA probe sets one or more PNA probesof claims 1-24 and at least one PNA probe for the analysis of S. aureus.

In one embodiment, the probes are differently labeled for independentanalysis of two or more Staphylococcus species.

In one aspect, provided herein are PNA probe sets comprising one or morePNA probes of claims 1-24 and at least one PNA blocking probe for theanalysis of one or more Staphylococcus species other than S. aureus.

In another embodiment, the probe sets further comprise a PNA blockingprobe for analysis of S. aureus.

In one aspect, provided herein are methods for the analysis ofStaphylococcus species other than S. aureus in a sample, comprising: a)contacting at least one of the PNA probes of claims 1-18 to the sample,b) hybridizing the PNA probe to a target sequence of Staphylococcusspecies other than S. aureus in the sample; and c) detecting thehybridization, wherein the detection of hybridization is indicative ofthe presence, identity and/or amount of Staphylococcus species otherthan S. aureus in the sample.

In one aspect, provided herein are methods for the analysis of two ormore Staphylococcus species, said method comprising: a) contacting a PNAprobe set of claims 1-18 to the sample, b) hybridizing the PNA probes toa target sequence of Staphylococcus species in the sample; and c)detecting the hybridization, wherein the detection of hybridization isindicative of the presence, identity and/or amount of Staphylococcusspecies in the sample.

In one embodiment, two or more Staphylococcus species are S. aureus andone or more Staphylococcus species other than S. aureus.

In one embodiment the probe sets are used for analysis of a cohort ofStaphylococcus species other than S. aureus.

In one embodiment, the cohort of Staphylococcus species other than S.aureus detected is Coagulase-Negative Staphylococci, clinicallysignificant Coagulase-Negative Staphylococci, or a subset of clinicallysignificant Coagulase-Negative Staphylococci.

In another embodiment, the probes are independently detectablenon-independently detectable, or a combination of independently andnon-independently detectable, wherein the probes differ from one anotherby as little as a single base, and are complementary or substantiallycomplementary to partially conserved target regions of phylogeneticallyrelated organisms.

In one embodiment, probes or probe sets are used to eliminate or reducecross hybridization between a Staphylococcus aureus specific probe, anda Staphylococcus scheiferi target.

In one embodiment, S. aureus and one or more Staphylococcus speciesother than S. aureus are simultaneously and independently detected.

In one embodiment, the analysis takes place in situ. In anotherembodiment, the analysis takes place by fluorescence in situhybridization.

In one embodiment, the methods are used to detect a nucleic acidcomprising a target sequence wherein said nucleic acid has beensynthesized or amplified in a reaction.

In one embodiment, preferred nucleic acid synthesis or nucleic acidamplification reactions are selected from the group consisting of:Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), StrandDisplacement Amplification (SDA), Transcription-Mediated Amplification(TMA), Rolling Circle Amplification (RCA) and Q beta replicase.

In one embodiment, the methods further comprise adding at least oneblocking probe to reduce or eliminate hybridization of the PNA probe tonon-target sequence.

In another embodiment, the target sequence is immobilized to a surface.In one embodiment, the PNA probe is immobilized to a surface. In oneembodiment, the PNA probe is one component of an array. In oneembodiment, the sample is a biological sample. In another embodiment,the biological sample is blood, urine, secretion, sweat, sputum, stool,mucous, or cultures thereof.

In one aspect, provided herein are kits suitable for performing an assayfor analysis of one or more Staphylococcus species other than S. aureusin a sample, wherein said kit comprises: a) a PNA probe according toclaim 1 to 24 and b) other reagents or compositions necessary to performthe assay.

In one embodiment, one or more Staphylococcus species other than S.aureus and at least one other microorganism optionally present in asample are independently detected, identified and/or quantitated.

In one embodiment, one or more Staphylococcus species other than S.aureus and S. aureus optionally present in a sample are independentlydetected, identified and/or quantitated.

In another embodiment, one or more Staphylococcus species other than S.aureus optionally present in a sample is detected, identified and/orquantitated and its susceptibility to antimicrobial agents isdetermined.

In one embodiment, one or more Staphylococcus species other than S.aureus detected is Coagulase-Negative Staphylococci, clinicallysignificant Coagulase- Negative Staphylococci, or a subset of clinicallysignificant Coagulase-Negative Staphylococci.

In another embodiment, the kit is used in an in situ hybridizationassay.

In one embodiment, the kit is a fluorescence in situ hybridization assayfor simultaneous, independent (multiplex) identification of one moreStaphylococcus species other than S. aureus and of S. aureus.

In one embodiment, the kit is used for a real-time PCR assay.

In one embodiment, the kit is used to examine clinical samples such asclinical specimens or cultures thereof.

Other embodiments are disclosed infra.

DETAILED DESCRIPTION OF THE INVENTION

1. Definitions:

As used herein, the term “nucleobase” means those naturally occurringand those non-naturally occurring heterocyclic moieties commonly knownto those who utilize nucleic acid technology or utilize peptide nucleicacid technology to thereby generate polymers that can sequencespecifically bind to nucleic acids.

As used herein, the term “nucleobase sequence” means any segment of apolymer that comprises nucleobase-containing subunits. Non-limitingexamples of suitable polymers or polymer segments includeoligodeoxynucleotides, oligoribonucleotides, peptide nucleic acids,nucleic acid analogs, nucleic acid mimics, and/or chimeras.

As used herein, the term “target sequence” means the nucleobase sequencethat is to be detected in an assay.

As used herein, the term “probe” means a polymer (e. g. a DNA, RNA, PNA,chimera or linked polymer) having a probing nucleobase sequence that isdesigned to sequence-specifically hybridize to a target sequence of atarget molecule of an organism of interest.

As used herein, the term “peptide nucleic acid” or “PNA” means anyoligomer, linked polymer or chimeric oligomer, comprising two or morePNA subunits (residues), including any of the polymers referred to orclaimed as peptide nucleic acids in U.S. Pat. Nos. 5,539,082, 5,527,675,5,623,049, 5,714,331, 5,736,336, 5,773,571, 5,786,461, 5,837,459,5,891,625, 5,972,610, 5,986,053, 6,107,470 and 6,357,163. In the mostpreferred embodiment, a PNA subunit consists of a naturally occurring ornon-naturally occurring nucleobase attached to the aza nitrogen of theN- [2-(aminoethyl)] glycine backbone through a methylene carbonyllinkage.

As used herein, the terms “label” and “detectable moiety” areinterchangeable and shall refer to moieties that can be attached to aprobe to thereby render the probe detectable by an instrument or method.

Reference herein to “Staphylococcus species other than S. aureus,” or arelated phrase means essentially one or more Staphylococcus species ofthe Staphylococcus genus except for S. aureus. With a few exceptions,Staphylococcus species other than S. aureus is equivalent tocoagulase-negative staphylococci, which is a medical expression forStaphylococcus species other than S. aureus. A cohort of Staphylococcusspecies other than S. aureus essentially means two of more species ofStaphylococcus species other than S. aureus and may comprise allcoagulase-negative staphylococci, clinically significantcoagulase-negative staphylococci or a subset of these. Staphylococcusspecies other than Staphylococcus aureus, may include, for example S.capitis, S.cohnii, S. epidermidis, S. saprophyticus, S. intermedius, S.hyicus, S. haemolyticus, S. hominis, S. Iugdunensis, S. saccharolyticus,S. schleiferi, S. sciuri, S. simulans, S. warneri and/or S. xylosus.

The term “sample” as used herein refers to any biological sample orclinical sample that could contain an analyte for detection. Preferablythe biological sample is in liquid form or as a tissue sample. Mostpreferably, the sample is from blood culture. Liquid samples includeclinical samples, e.g. urine, blood, wounds, sputum, laryngeal swabs,gastric lavage, bronchial washings, aspirates, serum, nasal discharge,sweat, plasma, semen, cerebrospinal fluid, tears, pus, amniotic fluid,saliva, lung aspirate, gastrointestinal contents, vaginal discharge,urethral discharge, expectorates and cultures thereof. Preferred tissuesamples or cultures thereof include chorionic villi specimens, skinepithelials, genitalia epithelials, gum epithelials, throat epithelials,hair and biopsies. Tissue samples may be either freshly prepared, orpreserved for some time in a fixative such as but not limited toethanol, methanol, paraformaldehyde, glutaraldehyde, formalin, paraffin,formaldehyde, formamide, or mixtures thereof. Non-clinical samplesinclude food, beverages, water, pharmaceutical products, personal careproducts, dairy products or environmental samples and cultures thereof.

As used herein, “multiplex assay” includes assays in which multipletargets can potentially be detected, and identified. Identification canbe specific, e.g., a particular species of microorganism, or generic,e.g., a particular genus of microorganism. Generic identification mayalso include the identification of a cohort of species ofmicroorganisms, which includes one or more members of a defined group ofspecies. One example of a defined group of species is thecoagulase-negative staphylococci.

As used herein, independent and simultaneous detection includes an assaythat can at once yield identification results for more than one target.For example, the detection of one or species of Staphylococcus otherthan aureus, and Staphylococcus aureus may be done at the same time withunique labels for each probe, thus resulting in independent andsimultaneous detection.

Other reagents or compositions necessary to perform the assay, mayinclude, for example, wash solutions, slides, coverslips, one or morePNA probes according to the invention, culture vessels, slide warmer,incubator, mounting fluid, and PCR components, including enzymes andbuffers.

PNA probes (originally described in U.S. Pat. No. 5,539,082 and Egholmet al., Nature 365:566 -568 (1993), herein attached as reference) haveinherent physico/chemical characteristics as compared to naturallyoccurring nucleic acid probes, which allow the design of rapid andaccurate assays. PNA probes offer another advantage over nucleic acidprobes when applied in fluorescence in situ hybridization (FISH) assaysdue to their improved cellular penetration of the rigid cell wall ofGram-positive bacteria such as Staphylococcus species. Where nucleicacid probes require fixation and permeabilization with cross-linkingagents and/or enzymes (for example see Kempf et al., J. Clin. Microbiol38:830-838 (2000)), PNA probes can be applied directly following smearpreparation as exemplified in Example 1.

In preferred embodiments, PNA probes have relatively short nucleobasesequences, such as 15 nucleobases as described in Example 1. Naturallyoccurring nucleic acid probes due to their weaker stabilities and lowermelting temperatures (Tm) are typically at least 18 nucleobases inlength (For example see Kempf et al., J. Clin. Microbiol 38:830-838(2000)). The greater specificity of PNA probes provides betterdiscrimination to closely related non-target sequences with a single orjust a few nucleobase difference(s) as required for analysis of rRNA orrDNA of closely related Staphylococcus species.

Exemplary PNA probe nucleobase sequences according to the inventioninclude: AGA-CGT-GCA-TAG-T (Seq. Id. No.1), GCT-AAT-ACG-GCG (Seq. Id.No. 2) and GCT-AAT-ACG-CCG-C (Seq. Id. No. 3).

In yet another embodiment, the PNA probes may be part of a PNA probe setcomprising either two or more PNA probes for analysis of two or morespecies of Staphylococcus species other than S. aureus or at least onePNA probe for analysis of Staphylococcus species other than S. aureusand a PNA probe for analysis of S. aureus. That is, some PNA probes ofthe invention are specific for two or more species of Staphylococcusspecies other then S. aureus. Preferably, PNA probes within a PNA probeset are differently labeled for independent analysis of two or moreStaphylococcus species. In a modification of this embodiment, and asexemplified in Examples 1 and 2, multiple probes may be identicallylabeled to detect a particular Staphylococcus species or cohort ofStaphylococcus species, while an optional other probe or probes isdifferently labeled for analysis of a second species or cohort ofspecies.

The method according to the invention comprises contacting a sample withone or more of the PNA probes described above. According to the method,the presence, absence and/or number of Staphylococcus species other thanStaphylococcus aureus are detected, identified and/or quantitated bycorrelating the hybridization, under suitable hybridization conditions,of the probing nucleobase sequence of the probe to the target sequence.Consequently, the analysis is based on a single assay with a definitiveoutcome. In contrast, current routine methods for definitive analysis ofStaphylococcus species other than S. aureus are based on multiplephenotypic characteristics involving multiple tests.

In another embodiment, the PNA probes are applied simultaneously withpreviously published PNA probes for analysis of Staphylococcus aureus(see Oliveira et al., J. Clin. Microbiol. 40:247-251 (2002)) either inparallel reactions or in the same reaction (multiplex) for simultaneousanalysis of Staphylococcus aureus and Staphylococcus species other thanS. aureus. This way the presence of Staphylococcus species other than S.aureus are further supported by the absence of S. aureus specific signalor visa versa, such final test results are interpreted on the basis onboth a positive and a negative reaction. The PNA probe set thereforeprovides internal controls which eliminate the need to perform separatecontrol experiments. Preferably, the two PNA probes are independentlylabeled such that the analysis is performed in one reaction (multiplex).Simultaneous analysis is also an advantage for specimens containing amixture of both S. aureus and Staphylococcus species other than S.aureus. In such cases the use of the PNA probes for FISH offers theadvantage of single cell detection, such that cells of bothStaphylococcus species other than S. aureus, and S. aureus can be viewedsimultaneously and differentiated by specific labels as exemplifiedbelow. In contrast other technologies are not able to distinguish mixedcultures from false-positive reactions without performing additionalcontrol experiments.

In still another embodiment, this invention is directed to kits suitablefor performing an assay that detects, identifies and/or quantitatesStaphylococcus species other than Staphylococcus aureus optionallypresent in a sample and/or determination of antibiotic resistance. Thekits of this invention comprise one or more PNA probes and otherreagents or compositions that are selected to perform an assay orotherwise simplify the performance of an assay. In particular, thecombined analysis of Staphylococcus species other than S. aureus andStaphylococcus aureus is well-suited for routine testing of GPCCpositive blood culture bottles where the use of multiple PNA probesserve secondarily as internal controls.

Another benefit derived from the use of multiple PNA probes is the useof blocking probes. In this preferred embodiment, the blocking probestrategy is employed in the design of probes for use in a multiplexassay where probes are directed against similar regions of conservedtarget molecules in closely related organisms. For example, a pair ofindependently detectable probes which are substantially similar innucleobase sequence and Tm may be designed to hybridize to highlyconserved regions of two targets in which the targeted nucleobasesequences differ by only one base. In this case, the tendency of thefirst probe to hybridize non-specifically to the complementary target ofthe second probe is discouraged by the presence and relative stabilityof the second probe/target hybrid. Similarly, multiple probes may bedesigned which are either independently detectable or non-independentlydetectable, or a combination of independently and non-independentlydetectable, which all differ from one another by as little as a singlebase, and which are complementary, or at least substantiallycomplementary to partially conserved target regions of phylogeneticallyrelated organisms. The competition for target sites can result in higherprobe specificities, while lowering the likelihood of crosshybridization.

Those of ordinary skill in the art will appreciate that a suitable PNAprobe need not have exactly these probing nucleobase sequences describedherein to be operative but may be modified according to the particularassay conditions. For example, shorter PNA probes can be prepared bytruncation of the nucleobase sequence if the stability of the hybridneeds to be modified to thereby lower the Tm and/or adjust forstringency. Similarly, the nucleobase sequence may be truncated at oneend and extended at the other end as long as the discriminatingnucleobases remain within the sequence of the PNA probe. Such variationsof the probing nucleobase sequences within the parameters describedherein are considered to be embodiments of this invention.

Those of ordinary skill in the art will also appreciate that thecomplement probing sequence is equally suitable for assays, such as butnot limited to real-time PCR, that are directed against rDNA as a targetsequence.

2. Description

I. General:

PNA Synthesis:

Methods for the chemical assembly of PNAs are well known (see: U.S. Pat.Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571,5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053 and 6,107,470).

PNA Labeling:

Preferred non-limiting methods for labeling PNAs are described in U.S.Pat. No. 6,110,676, 6,361,942, and 6,355,421, the examples section ofthese specifications or are otherwise well known in the art of PNAsynthesis and peptide synthesis.

Labels:

Non-limiting examples of detectable moieties (labels) suitable forlabeling PNA probes used in the practice of this invention would includea dextran conjugate, a branched nucleic acid detection system, achromophore, a fluorophore, a spin label, a radioisotope, an enzyme, ahapten, an acridinium ester and a chemiluminescent compound.

Other suitable labeling reagents and preferred methods of attachmentwould be recognized by those of ordinary skill in the art of PNA,peptide or nucleic acid synthesis.

Preferred haptens include 5 (6)-carboxyfluorescein, 2,4-dinitrophenyl,digoxigenin, and biotin.

Preferred fluorochromes (fluorophores) include 5 (6)-carboxyfluorescein(Flu), tetramethyl-6-carboxyrhodamine (tamra),6-((7-amino-4-methylcoumarin-3-acetyl) amino) hexanoic acid (Cou), 5(and 6)-carboxy-X- rhodamine (Rox), Cyanine 2 (Cy2) Dye, Cyanine 3 (Cy3)Dye, Cyanine 3.5 (Cy3.5) Dye, Cyanine 5 (Cy5) Dye, Cyanine 5.5 (Cy5.5)Dye Cyanine 7 (Cy7) Dye, Cyanine 9 (Cy9) Dye (Cyanine dyes 2, 3, 3.5, 5and 5.5 are available as NHS esters from Amersham, Arlington Heights,Ill.), JOE, Tamara or the Alexa dye series (Molecular Probes, Eugene,Oreg.).

Preferred enzymes include polymerases (e. g. Taq polymerase, Klenow PNApolymerase, T7 DNA polymerase, Sequenase, DNA polymerase 1 and phi29polymerase), alkaline phosphatase (AP), horseradish peroxidase (HRP) andmost preferably, soy bean peroxidase (SBP).

Unlabeled Probes:

The probes that are used for the practice of this invention need not belabeled with a detectable moiety to be operable within the methods ofthis invention, for example, the probes of the invention may be attachedto a solid support, which renders them detectable by known arraytechnologies.

Self-indicating Probes:

Beacon probes are examples of self-indicating probes which include adonor moiety and an acceptor moiety. The donor and acceptor moietiesoperate such that the acceptor moieties accept energy transferred fromthe donor moieties or otherwise quench signal from the donor moiety.Though the previously listed fluorophores (with suitable spectralproperties) might also operate as energy transfer acceptors, preferably,the acceptor moiety is a quencher moiety. Preferably, the quenchermoiety is a non-fluorescent aromatic or heteroaromatic moiety. Thepreferred quencher moiety is 4-((-4-(dimethylamino) phenyl) azo) benzoicacid (dabcyl). In a preferred embodiment, the self-indicating Beaconprobe is a PNA Linear Beacon as more fully described in U.S. Pat. No.6,485,901.

In another embodiment, the self-indicating probes of this invention areof the type described in WIPO patent application W097/45539. Theseself-indicating probes differ as compared with Beacon probes primarilyin that the reporter must interact with the nucleic acid to producesignal.

Spacer/Linker Moieties:

Generally, spacers are used to minimize the adverse effects that bulkylabeling reagents might have on hybridization properties of probes.Preferred spacer/linker moieties for the nucleobase polymers of thisinvention consist of one or more aminoalkyl carboxylic acids(e.g.,aminocaproic acid), the side chain of an amino acid (e.g., theside chain of lysine or omithine), natural amino acids (e.g., glycine),aminooxyalkylacids (e.g., 8-amino-3,6-dioxaoctanoic acid), alkyl diacids(e.g., succinic acid), alkyloxy diacids (e.g., diglycolic acid) oralkyldiamines (e.g., 1,8-diamino-3,6-dioxaoctane).

Hybridization Conditions/Stringency:

Those of ordinary skill in the art of nucleic acid hybridization willrecognize that factors commonly used to impose or control stringency ofhybridization include formamide concentration (or other chemicaldenaturant reagent), salt concentration (i.e., ionic strength),hybridization temperature, detergent concentration, pH and the presenceor absence of chaotropes. Optimal stringency for a probe/target sequencecombination is often found by the well-known technique of fixing severalof the aforementioned stringency factors and then determining the effectof varying a single stringency factor. The same stringency factors canbe modulated to thereby control the stringency of hybridization of a PNAto a nucleic acid, except that the hybridization of a PNA is fairlyindependent of ionic strength. Optimal stringency for an assay may beexperimentally determined by examination of each stringency factor untilthe desired degree of discrimination is achieved.

Suitable Hybridization Conditions:

Generally, the more closely related the background causing nucleic acidcontaminants are to the target sequence, the more carefully stringencymust be controlled. Blocking probes may also be used as a means toimprove discrimination beyond the limits possible by optimization ofstringency factors. Suitable hybridization conditions will thus compriseconditions under which the desired degree of discrimination is achievedsuch that an assay generates an accurate (within the tolerance desiredfor the assay) and reproducible result.

Aided by no more than routine experimentation and the disclosureprovided herein, those of skill in the art will easily be able todetermine suitable hybridization conditions for performing assaysutilizing the methods and compositions described herein. Suitable insitu hybridization or PCR conditions comprise conditions suitable forperforming an in situ hybridization or PCR procedure. Thus, suitable insitu hybridization or PCR conditions will become apparent to those ofskill in the art using the disclosure provided herein, with or withoutadditional routine experimentation.

Blocking Probes:

Blocking probes are nucleic acid or non-nucleic acid probes that can beused to suppress the binding of the probing nucleobase sequence of theprobing polymer to a non-target sequence. Preferred blocking probes arePNA probes (see: U.S. Pat. No. 6,110,676). It is believed that blockingprobes operate by hybridization to the non-target sequence to therebyform a more thermodynamically stable complex than is formed byhybridization between the probing nucleobase sequence and the non-targetsequence. Through formation of the more stable and preferred complex,the less stable non-preferred complex between the probing nucleobasesequence and the non-target sequence is prevented from forming. Thus,blocking probes can be used with the methods, kits and compositions ofthis invention to suppress the binding of the probes to a non-targetsequence that might be present and interfere with the performance of theassay.

Probing Nucleobase Sequence:

The probing nucleobase sequence of a probe of this invention is thespecific sequence recognition portion of the construct. Therefore, theprobing nucleobase sequence is a nucleobase sequence designed tohybridize to a specific target sequence wherein the presence, absence oramount of the target sequence can be used to directly or indirectlydetect the presence, absence or number of organisms of interest in asample. Consequently, with due consideration to the requirements of aprobe for the assay format chosen, the length and sequence compositionof the probing nucleobase sequence of the probe will generally be chosensuch that a stable complex is formed with the target sequence undersuitable hybridization conditions.

The preferred nucleobase sequences of the probes of this invention foranalysis of Staphylococcus species other than S. aureus are:AGA-CGT-GCA-TAG-T (Seq. ID. No. 1), GCT-AAT-ACG-GCG (Seq. ID. No. 2),GCT-AAT-ACG-CCG-C (Seq. ID. No. 3), and the complements thereof.

This invention contemplates that variation in these identified probingnucleobase sequences shall also provide probes that are suitable for theanalysis of Staphylococcus species other than S. aureus. Such variationof the probing nucleobase sequences within the parameters describedherein are considered to be an embodiment of this invention. Commonvariations include, deletions, insertions and frame shifts.Additionally, a shorter probing nucleobase sequence can be generated bytruncation of the sequences identified above.

A probe of this invention will generally have a probing nucleobasesequence that is exactly complementary to the target sequence.Alternatively, a substantially complementary probing nucleobase sequencemight be used since it has been demonstrated that greater sequencediscrimination can be obtained when utilizing probes wherein thereexists one or more point mutations (base mismatch) between the probe andthe target sequence (See: Guo et al., Nature Biotechnology 15:331-335(1997)). Consequently, the probing nucleobase sequence may be only asmuch as 86% homologous to the probing nucleobase sequences identifiedabove. Substantially complementary probing nucleobase sequences withinthe parameters described above are considered to be an embodiment ofthis invention.

Complements of the probing nucleobase sequence are considered to be anembodiment of this invention, since it is possible to generate asuitable probe if the target sequence to be detected has been amplifiedor copied to thereby generate the complement to the identified targetsequence.

Detection, Identification and/or Enumeration:

By detection is meant analysis for the presence or absence of theorganism optionally present in the sample. By identification is meantestablishment of the identity of the organism by genus name, by genusand species name, or by other suitable category which serves to classifythe organism(s) of interest. By quantitation is meant enumeration of theorganisms in a sample. Some assay formats provide simultaneousdetection, identification and enumeration (for example see Stender, H.et al., J. Microbiol. Methods. 45:31-39 (2001), others provide detectionand identification (for example see Stender, H. et al., Int. J. Tuberc.Lung Dis. 3:830-837 (1999) and yet other assay formats just provideidentification (for example see Oliveira, K et al. J. Clin. Microbiol.40:247-251 (2002)).

Independent and Simultaneous Detection:

In a preferred embodiment of this invention, a multiplex assay isdesigned to detect one target with a labeled probe, while simultaneouslydetecting another target with a differently labeled probe. In a morepreferred embodiment of the invention, the targets constitute rRNA orrDNA from two or more Staphylococcus species. In the most preferredembodiment, the assay is a PNA FISH assay designed to detect one orspecies of Staphylococcus other than aureus, and Staphylococcus aureus.

Antibiotic Resistance

By determination of resistance to antibiotics is meant analysis of anorganism's susceptibility to antibiotics based on specific genes or geneproducts, or mutations associated with resistance or susceptibility toantimicrobial agents.

II. Preferred Embodiments of the Invention:

a. PNA Probes:

In one embodiment, this invention is directed to PNA probes. The PNAprobes of this invention are suitable for detecting, identifying and/orquantitating Staphylococcus species other than S. aureus optionallypresent in a sample. General characteristics (e.g., length, labels,nucleobase sequences, linkers, etc.) of PNA probes suitable for theanalysis have been previously described herein. The preferred probingnucleobase sequence of PNA probes of this invention are listed in Table1.

TABLE 1 1 2 A Sequence ID Nucleobase sequence B Seq. ID. No. 1AGA-CGT-GCA-TAG-T C Seq. ID. No. 2 GCT-AAT-ACG-GCG D Seq. ID. No. 3GCT-AAT-ACG-CCG-C

The PNA probes of this invention may comprise only a probing nucleobasesequence (as previously described herein) or may comprise additionalmoieties. Non-limiting examples of additional moieties includedetectable moieties (labels), linkers, spacers, natural or non-naturalamino acids, or other subunits of PNA, DNA or RNA. Additional moietiesmay be functional or non-functional in an assay. Generally however,additional moieties will be selected to be functional within the designof the assay in which the PNA probe is to be used. The preferred PNAprobes of this invention are labeled with one or more detectablemoieties selected from the group consisting of fluorophores, enzymes andhaptens.

In preferred embodiments, the probes of this invention are used in insitu hybridization (ISH) and fluorescence in situ hybridization assays.Excess probe used in an ISH or FISH assay typically must be removed sothat the detectable moiety of the specifically bound probe can bedetected above the background signal that results from still present butunhybridized probe. Generally, the excess probe is washed away after thesample has been incubated with probe for a period of time. However, theuse of self-indicating probes is a preferred embodiment of thisinvention, since there is no requirement that excess self-indicatingprobe be completely removed (washed away) from the sample since itgenerates little or no detectable background. In addition to ISH or FISHassays, self-indicating probes comprising the selected probingnucleobase sequence described herein are particularly useful in allkinds of homogeneous assays such as in real-time PCR or useful withself-indicating devices (e. g. lateral flow assay) or self-indicatingarrays.

b. PNA Probe Sets

Probe sets of this invention comprise two or more PNAs. In oneembodiment, some of the PNA probes of the set can be blocking probes. Inother embodiments, the probe set can be used to analyze two of moreStaphylococcus species other than S. aureus, or for the analysis of oneor more Staphylococcus species other than S. aureus, and S. aureus.

c. Methods:

In another embodiment, this invention is directed to a method suitablefor analysis of Staphylococcus species other than S. aureus optionallyin a sample. The general and specific characteristics of PNA probessuitable for the analysis of Staphylococcus species other than S. aureushave been previously described herein. Preferred probing nucleobasesequences are listed in Table 1.

The method for analysis of Staphylococcus species other than S. aureusin a sample comprises contacting the sample with one or more PNA probessuitable for hybridization to a target sequence which is specific toStaphylococcus species other than S. aureus. According to the method,Staphylococcus species other than S. aureus in the sample is thendetected, identified and/or quantitated or its resistance to antibioticsis determined. This is made possible by correlating hybridization, undersuitable hybridization conditions or suitable in situ hybridizationconditions, of the probing nucleobase sequence of a PNA probe to thetarget sequence of Staphylococcus species other than S. aureus sought tobe detected with the presence, absence or number of the Staphylococcusspecies other than S. aureus organisms in the sample. Typically, thiscorrelation is made possible by direct or indirect detection of theprobe/target sequence hybrid. In a preferred embodiment, a PNA probe setis used for simultaneous analysis of Staphylococcus species other thanS. aureus and S. aureus using differently labeled PNA probes.

Fluorescence in situ Hybridization and Real-time PCR:

The PNA probes, methods, kits and compositions of this invention areparticularly useful for the rapid probe-based analysis of Staphylococcusspecies other than S. aureus, preferably using PNA probe sets forsimultaneous analysis of two or more Staphylococcus species. Inpreferred embodiments, in situ hybridization or PCR is used as the assayformat for analysis of Staphylococcus species other than S. aureus. Mostpreferably, fluorescence in situ hybridization (PNA FISH) or real-timePCR is the assay format (Reviewed by Stender et al. J. Microbiol.Methods 48:1-17 (2002)). Preferably, smears for PNA FISH analysis arenot treated with cross-linking agents or enzymes prior to hybridization.

In one embodiment, the method includes synthesizing a nucleic acid froma sample, for example by nucleic acid amplification. Preferred nucleicacid amplification reactions are selected from the group consisting of:Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), StrandDisplacement Amplification (SDA), Transcription-Mediated Amplification(TMA), Rolling Circle Amplification (RCA) and Q beta replicase.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.). Methods and techniquesapplicable to polymer (including protein) array synthesis have beendescribed in U.S. Ser. No. 09/536,841, WO 00/58516, U.S. Pat. Nos.5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783,5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215,5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734,5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324,5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860,6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCTApplications Nos. PCT/US99/00730 (International Publication Number WO99/36760) and PCT/USO1/04285, which are all incorporated herein byreference in their entirety for all purposes. Patents that describesynthesis techniques in specific embodiments include U.S. Pat. Nos.5,412,087, 6,147,205, 6,262,216, 6,310,189, 5,889,165, and 5,959,098.For PCR methods see, e.g., PCR Technology: Principles and Applicationsfor DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992);PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al.,Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic AcidsRes. 19, 4967 (1991); Eckert et al., PCR Methods and Applications 1,17(1991); PCR (Eds. McPherson et al., IRL Press, Oxford); and U.S. Pat.Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188, and 5,333,675, and eachof which is incorporated herein by reference in their entireties for allpurposes. The sample may be amplified on the array. See, for example,U.S. Pat. No. 6,300,070 and U.S. patent application Ser. No. 09/513,300,which are incorporated herein by reference. Nucleic acid arrays that areuseful in the present invention include those that are commerciallyavailable from Affymetrix (Santa Clara, Calif.). Other suitableamplification methods include the ligase chain reaction (LCR) (e.g., Wuand Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077(1988) and Barringer et al. Gene 89:117 (1990)), transcriptionamplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989)and WO88/10315), self-sustained sequence replication (Guatelli et al.,Proc. Nat. Acad. Sci. USA, 87,1874 (1990) and WO90/06995), selectiveamplification of target polynucleotide sequences (U.S. Pat. No.6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR)(U.S. Pat. No. 4,437,975), arbitrarily primed polymerase chain reaction(AP-PCR) (U.S. Pat. No. 5,413,909, 5,861,245) and nucleic acid basedsequence amplification (NABSA, see, U.S. Pat. Nos. 5,409,818, 5,554,517,and 6,063,603, each of which is incorporated herein by reference). Otheramplification methods that may be used are described in, U.S. Pat. Nos.5,242,794, 5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317, each ofwhich is incorporated herein by reference.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11,1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 and U.S.patent application Ser. Nos. 09/916,135, 09/920,491, 09/910,292, and10/013,598.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (2.sup.nd Ed.Cold Spring Harbor, N.Y., 1989); Berger and Kimmel Methods inEnzymology, Vol. 152, Guide to Molecular Cloning Techniques (AcademicPress, Inc., San Diego, Calif., 1987); Young and Davism, P.N.A.S,80:1194 (1983). Methods and apparatus for carrying out repeated andcontrolled hybridization reactions have been described in U.S. Pat. Nos.5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of whichare incorporated herein by reference.

Exemplary Assay Formats:

Exemplary methods for performing PNA FISH can be found in: Oliveira etal., J. Clin. Microbiol 40:247-251 (2002), Rigby et al., J. Clin.Microbiol. 40:2182-2186 (2002), Stender et al., J. Clin. Microbiol.37:2760-2765 (1999), Perry-O'Keefe et al., J. Microbiol. Methods47:281-292 (2001). According to one method, a smear of the sample, suchas, but not limited to, a positive blood culture, is prepared onmicroscope slides and covered with one drop of the fluorescent-labeledPNA probe in hybridization buffer. A coverslip is placed on the smear toensure even coverage, and the slide is subsequently placed on a slidewarmer or incubator at 55° C. for 90 minutes. Following hybridization,the coverslip is removed by submerging the slide into a pre-warmedstringent wash solution and the slide is washed for 30 minutes. Thesmear is finally mounted with one drop of mounting fluid, covered with acoverslip and examined by fluorescence microscopy.

Staphylococcus species other than S. aureus optimally present in asample which may be analyzed with the PNA probes contained in the kitsof this invention can be evaluated by several instruments, such as butnot limited to the following examples: microscope (for example seeOliveira et al., J. Clin. Microbiol 40:247-251 (2002)), radiationsensitive film (for example see Perry-O'Keefe et al., J. Appl.Microbiol. 90:180-189) (2001), camera and instant film (for example seeStender et al., J. Microbiol. Methods 42:245-253 (2000)), luminometer(for example see Stender et al., J. Microbiol. Methods. 46:69-75 (2001),laser scanning device (for example see Stender et al., J. Microbiol.Methods. 45: 31-39 (2001) or flow cytometer (for example see Wordon etal., Appl. Environ. Microbiol. 66:284-289 (2000)). Automated slidescanners and flow cytometers are particularly useful for rapidlyquantitating the number of microorganisms present in a sample ofinterest.

Exemplary methods for performing real-time PCR using self-reporting PNAprobes can be found in: Fiandaca et al., Abstract, Nucleic Acid-Basedtechnologies, DNA/RNA/PNA Diagnostics, Washington, DC, May 14-16, 2001,and Perry-O'Keefe et al., Abstract, International Conference on EmergingInfectious Diseases, Atlanta, 2002.

d. Kits:

In yet another embodiment, this invention is directed to kits suitablefor performing an assay, which analyzes Staphylococcus species otherthan S. aureus optionally present in a sample. The general and preferredcharacteristics of PNA probes suitable for the analysis ofStaphylococcus species other than S. aureus have been previouslydescribed herein. Preferred probing nucleobase sequences are listed inTable 1. Furthermore, methods suitable for using PNA probes to analyzeStaphylococcus species other than S. aureus in a sample have beenpreviously described herein.

The kits of this invention comprise one or more PNA probes and otherreagents or compositions which are selected to perform an assay orotherwise simplify the performance of an assay used to analyzeStaphylococcus species other than S. aureus in a sample. In preferredembodiments, the kit comprises a PNA probe set for simultaneous analysisof Staphylococcus species other than S. aureus and S. aureus usingindependently detectable PNA probes.

e. Exemplary Applications for Using the Invention:

The PNA probes, methods and kits of this invention are particularlyuseful for the analysis of Staphylococcus species other than S. aureusin clinical samples, e.g., urine, blood, wounds, sputum, laryngealswabs, gastric lavage, bronchial washings, biopsies, aspirates,expectorates as well as in food, beverages, water, pharmaceuticalproducts, personal care products, dairy products or environmentalsamples and cultures thereof. In preferred embodiments, the PNA probesare applied in a PNA probe set also containing a PNA probe for analysisof S. aureus.

Having described the preferred embodiments of the invention, it will nowbecome apparent to one of skill in the art that other embodimentsincorporating the concepts described herein may be used. It is felt,therefore, that these embodiments should not be limited to disclosedembodiments but rather should be limited only by the spirit and scope ofthe following claims.

EXAMPLE 1 Evaluation of Probe Sets

Set PNA probe (Seq. ID. No.) Sequence A. CNS16S09b/txr (Seq. ID. No. 1)TXR-OO-AGA-CGT- GCA-TAG-T B. CNS16S16g/txr (Seq. ID. No. 2)TXR-OO-GCT-AAT- ACG-GCG (Note: Conventional nomenclature used toillustrate the termini of the PNA probe; O = 8-amino-3,6-dioxaoctanoicacids; TXR = Texas Red.)

Bacterial Strains

Overnight cultures of reference strains (American Type CultureCollection, (ATCC) Manassas, Va., or Agricultural Research ServiceCulture Collection (NRRL) Peoria, Ill.) of Staphylococcus species wereprepared, including Staphylococcus epidermidis, Staphylococcus aureusand other relevant species were prepared.

Preparation of Smears.

For each strain, smears were prepared on a 8-mm diameter well of ateflon-coated microscope slide (AdvanDx, Woburn, Mass.) by mixing onedrop of culture with one drop of phosphate-buffered saline containing 1%(v/v) Triton X-100. The slide was then placed on a 55° C. slide warmerfor 20 min at which point the smears were dry. Subsequently, the smearswere disinfected by immersion into 96% (v/v) ethanol for 5-10 minutesand air-dried.

Fluorescence in situ hybridization (FISH).

Smears were covered with a drop of hybridization solution containing 10%(w/v) dextran sulfate, 10 mM NaCl, 30% (v/v) formamide, 0.1% (w/v)sodium pyrophosphate, 0.2% (w/v) polyvinylpyrrolidone, 0.2% (w/v)ficoll, 5 mM Na2EDTA, 1% (v/v) Triton X-100, 50 mM Tris/HCl pH 7.5 and250 nM CNS16S09b/txr (Set A) or CNS16S16g/txr (Set B). Coverslips wereplaced on the smears to ensure even coverage with hybridizationsolution, and the slides were subsequently placed on a slide warmer(Slidemoat, Boekel, Feasterville, Pa.) and incubated for 90 min at 55°C. Following hybridization, the coverslips were removed by submergingthe slides into approximately 20 ml/slide pre-warmed 25 mM Tris, pH 10,137 mM NaCl, 3 mM KCl in a water bath at 55° C. and washed for 30 min.Each smear was finally mounted using one drop of Mounting medium(AdvanDx, Woburn, Mass.) and covered with a coverslip. Microscopicexamination was conducted using a fluorescence microscope equipped witha FITC/Texas Red dual band filter set. Staphylococcus species other thanS. aureus was identified by red fluorescent cocci.

TABLE 2 1 2 3 4 A Species ID# Set A Set B B Candida albicans NRRL − −Y-17968 C Candida glabrata ATCC 15126 − − D Escherichia coli ATCC 43888− − E Klebsiella oxytoca ATCC 43086 − − F Klebsiella pneumoniae ATCC10031 − − G Pseudomonas aeruginosa ATCC 10145 − − H Enterococcusfaecalis ATCC 19433 − − I Enterococcus faecium ATCC 27270 − − JMicrococcus luteus ATCC 4698 − − K Staphylococcus aureus ATCC 11632 − −L Staphylococcus epidermidis ATCC 14990 Red + − M Staphylococcusgallinarum ATCC 700401 − Red + N Staphylococcus haemolyticus ATCC 29970Red + Red + O Staphylococcus lugdunensis ATCC 49576 − Red + PStaphylococcus saprophyticus ATCC 15305 Weak Red + Red + QStaphylococcus simulans ATCC 27851 − − R Staphylococcus schleiferi ATCC43808 Weak Red + Red + S Streptococcus agalactiae ATCC 13813 − − TStreptococcus pyogenes ATCC 12384 − −

With reference to Table 2, the table displays the data from Example 1,with species identification in column 1, species ID# in column 2, probeSet A results in column 3, and probe Set B results in column 4, asindicated by the header row, row A. The symbol “−”indicates a negativeresult, the symbol “+” indicates a positive result. Positive results arescored by observation of fluorescent cells in the sample, eitherfluorescent Red or Green. With reference to Table 2, column 3, resultsfor probe Set A, all species tested negative (−), except Staphylococcusepidermidis and Staphylococcus haemolyticus (rows L and N), which testedpositive, with Red fluorescence (“Red positive”), and Staphylococcussaprophyticus and Staphylococcus schleiferi (rows P and R) which wereboth weakly positive with red fluorescence. With reference to Table 2,column 4, results for probe Set B, all species tested negative (−),except Staphylococcus gallinarium, Staphylococcus haemolyticus,Staphylococcus lugdunensis, Staphylococcus saprophyticus, andStaphylococcus schleiferi (rows M-P and R), which tested Red positive.Thus, the example demonstrates that probe Sets A and B are both usefulfor detection of certain Staphylococcus species other than S. aureus,although weak signals are observed with certain species, and neitherprobe set is useful for detection of all Staphylococcus species otherthan S. aureus. It is worthwhile to note that Staphylococcus aureus, andseveral other Gram-positive cocci, such as Enterococcus faecalis, andStreptococcus pyogenes had negative results with both probe sets.

EXAMPLE 2 Evaluation of Probe Sets

Set PNA probe (Seq. ID. No.) Sequence C. CNS16S09b/txr (Seq. ID. No. 1)TXR-OO-AGA-CGT- GCA-TAG-T CNS16S16g/txr (Seq. ID. No. 2) TXR-OO-GCT-AAT-ACG-GCG Sau16S03/flu N/A Flu-OO-GCT-TCT- CGT-CCGTTC D. Ssim16S01/txr(Seq. ID. No. 3) TXR-OO-GCT-AAT- ACG-CCG-C E. CNS16S09b/txr (Seq. ID.No. 1) TXR-OO-AGA-CGT- GCA-TAG-T CNS16S16g/txr (Seq. ID. No. 2)TXR-OO-GCT-AAT- ACG-GCG Sau16S03/flu N/A Flu-OO-GCT-TCT- CGT-CCGTTCSsim16S01/txr (Seq. ID. No. 3) TXR-OO-GCT-AAT- ACG-CCG-C (Note:Conventional nomenclature used to illustrate the termini of the PNAprobe; O = 8-amino-3,6-dioxaoctanoic acids; TXR = Texas Red; Flu= carboxy-fluorescein.)

The probe Sets were tested exactly as described in Example 1, thoughtesting was performed on a partially different group of strains.

TABLE 3 1 2 3 4 5 A Species ATCC# Set C Set D Set E B Enterococcusfaecalis 29212 — — — C Enterococcus faecium 27270 — — — D Escherichiacoli 11229 — — — E Klebsiella pneumoniae 10031 — — — F Pseudomonasaeruginosa 10145 — — — G Staphylococcus aureus 11632 Green+ — Green+ HStaphylococcus capitis 35661 Red+ — Red+ I Staphylococcus epidermidis14990 Red+ — Red+ J Staphylococcus gallinarum 700401 Red+ — Red+ KStaphylococcus haemolyticus 29970 Red+ — Red+ L Staphylococcuslugdunensis 49576 Red+ — Red+ M Staphylococcus saprophyticus 15305 Red+— Red+ N Staphylococcus schleiferi 43808 Red+ — Red+ O Staphylococcussciuri 29061 Red+ — Red+ P Staphylococcus simulans 27851 — Red+ Red+ QStaphylococcus warneri 49454 Red+ — Red+ R Streptococcus agalactiae13813 — — — S Streptococcus pyogenes 29212 — — —

Probe Set C is a mixture of the probes from probe Sets A and B ofExample 1, combined with a third S. aureus specific probe, labeled withfluorescein. With reference to Table 3, the table displays the data fromExample 2, with species identification in column 1, species ATCC# incolumn 2, probe Set C results in column 3, probe Set D results in column4, and probe Set E results in column 5, as indicated by the header row,row A. The symbol “−”indicates a negative result, the symbol“+”indicates a positive result. Positive results are scored byobservation of fluorescent cells in the sample, either fluorescent Redor Green. With reference to Table 3, column 3, results for probe Set C,S. aureus tested Green positive. All other samples were negative forGreen fluorescence. All other Staphylococcus strains except S. simulanstested Red positive with probe Set C. No non-staphylococci tested Redpositive, or Green positive with probe Set C. With reference to column4, probe Set D, the only positive result was observed with S. simulanswhich was Red positive. Probe Set D contains a single Texas Red labeledprobe species, and thus it is not surprising that no green fluorescencewas observed. Probe Set E seen in Table 3, column 5 is a mixture of theprobes from probe Set C and D. In column 5, all Staphylococcus speciesare detected with either Green (S. aureus only) or Red (Staphylococcusspecies other than S. aureus) fluorescence, while no non-staphylococciare detected with fluorescent signals.

It has been demonstrated in previous experiments that the Sau16S03 probeforms a stabile hybrid with S.schleiferi even though the probe andtarget sequence are not perfectly complementary (Oliveira et al., J.Clin. Microbiol. 40:247-251 (2002)). In this example, the signal from S.schleiferi with probe Sets C and E is a Red positive. Thus, the use ofprobe Sets C or E provides a more specific detection of S. aureus withthe Sau16S03 probe, than use of the Sau16S03 probe on its own, aspreviously reported.

These data demonstrate that a probe mixture can be made which detects acohort of species by one fluorescent label, and a single species with asecond fluorescent label. Though the Staphylococcus species tested inthis experiment are considered to be the most clinically relevant, otherexperiments not included here demonstrated that many otherStaphylococcus species can be detected with probe Sets A, B, C and E.

All references cited herein, whether in print, electronic, computerreadable storage media or other form, are expressly incorporated byreference in their entirety, including but not limited to, abstracts,articles, journals, publications, texts, treatises, technical datasheets, internet web sites, databases, patents, patent applications, andpatent publications.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A PNA probe comprising a nucleobase sequence suitable for theanalysis of one or more Staphylococcus species other than S. aureus. 2.A PNA probe comprising a nucleobase sequence suitable for the analysisof two or more Staphylococcus species other than S. aureus.
 3. The PNAprobe of claims 1-2, wherein a target sequence of the Staphylococcusspecies comprises rRNA, rDNA or a complement of rRNA or rDNA.
 4. The PNAprobe of claim 1-2, wherein the nucleobase sequence suitable for theanalysis of Staphylococcus epidermidis, simulans, haemolyticus,lugdunensis, or saprophyticus, comprises one or more of a PNA probecomplementary to a target sequence of the Staphylococcus epidermidissimulans, haemolyticus, lugdunensis, or saprophyticus rRNA or rDNA or acomplement thereof.
 5. The PNA probe of claim 1, wherein the nucleobasesequence suitable for the analysis of Staphylococcus epidermidis,simulans, haemolyticus, lugdunensis, or saprophyticus and one or moreother Staphylococcus species other than Staphylococcus aureus, comprisesa PNA probe complementary to a target sequence of Staphylococcusepidermidis, simulans, haemolyticus, lugdunensis, or saprophyticus rRNAor rDNA or a complement thereof.
 6. The PNA probe of claims 1, 2 or 5,wherein at least a portion of the probe is at least about 86% identicalto the nucleobase sequence or complement thereof selected from thefollowing sequences: AGA-CGT-GCA-TAG-T (SEQ ID NO: 1), GCT-AAT-ACG-GCG(SEQ ID NO: 2), GCT-AAT-ACG-CCG-C (SEQ ID NO: 3).
 7. The PNA probe ofclaims 1, 2 or 5, wherein at least a portion of the probe is selectedfrom the following sequences: AGA-CGT-GCA-TAG-T (SEQ ID NO:- 1),GCT-MT-ACG-GCG (SEQ ID NO: 2), GCT-AAT-ACG-CCG-C( SEQ ID NO: 3).
 8. ThePNA probe of claims 1, 2, or 5, wherein the probe sequence is betweenabout 8 and about 17 subunits in length.
 9. The PNA probe of claims 1,2, or 5, wherein the probe is labeled with at least one detectablemoiety.
 10. A PNA probe set comprising one or more PNA probes of claims1 or 2, and at least one PNA probe for the analysis of S. aureus.
 11. APNA probe set comprising one or more PNA probes of claims 1 or 2, and atleast one PNA blocking probe for the analysis of one or moreStaphylococcus species other than S. aureus.
 12. A method for theanalysis of Staphylococcus species other than S. aureus in a sample,comprising: a) contacting at least one of the PNA probes of claims 1-9to the sample, b) hybridizing the PNA probe to a target sequence ofStaphylococcus species other than S. aureus in the sample; and c)detecting the hybridization, wherein the detection of hybridization isindicative of the presence, identity and/or amount of Staphylococcusspecies other than S. aureus in the sample.
 13. A method for theanalysis of two or more Staphylococcus species, said method comprising:a) contacting a PNA probe set to the sample, b) hybridizing the PNAprobes to a target sequence of Staphylococcus species in the sample; andc) detecting the hybridization, wherein the detection of hybridizationis indicative of the presence, identity and/or amount of Staphylococcusspecies in the sample.
 14. The method of claim 12 or 13, wherein theprobes are independently detectable non-independently detectable, or acombination of independently and non-independently detectable, whereinthe probes differ from one another by as little as a single base, andare complementary or substantially complementary to partially conservedtarget regions of phylogenetically related organisms.
 15. The method ofclaim 12 or 13, wherein S. aureus and one or more Staphylococcus speciesother than S. aureus are simultaneously and independently detected. 16.A kit suitable for performing an assay for analysis of one or moreStaphylococcus species other than S. aureus in a sample, wherein saidkit comprises: a) a PNA probe according to claim 1 to 9 and b) otherreagents or compositions necessary to perform the assay.